K85 and K297 were required for HERC4-mediated c-Maf ubiquitination. (A) Lysine residues in c-Maf protein. Each K was changed to arginine (R) by site-direct mutagenesis. (B) Wild-type (WT) and mutant c-Maf plasmids were transfected into HEK293 cells with or without a HERC4 plasmid. Forty-eight hours later, cell lysates were subjected to an IB assay for c-Maf. (C) WT or all-lysine-substituted (K0) c-Maf plasmids were transfected with HERC4 and Ub-Flag. Forty-eight hours later, cells were treated with MG132 for 2 hours before cell lysate preparation. Cell lysates were immunoprecipitated with an anti–c-Maf antibody, followed by IB against Flag (Ub) or c-Maf. (D) HEK293 cells were transfected with HERC4, Flag-Ub, and WT or the indicated c-Maf variant. Forty-eight hours later, cells were treated with or without MG132 before cell lysate preparation. Cell lysates were then subjected to IP with an anti-HA antibody (for c-Maf), and a subsequent IP assay against Flag (for Ub).